Figure 6

Introducing Morpholinos into young Xenopus tadpoles by electroporation and in vitro approaches. a-d: Frontal sections of embryos 24 h after electroporation with lissamine-tagged MO. Large numbers of cells can be loaded with MO in both the brain (a) and the eye (c). Microanatomy of both structures appears normal (b and d). e-f: Co-electroporation of pCS2GAP-GFP with lissamine-tagged special delivery MO. e: A higher magnification image of a co-electroporated brain. The MO signal was de-saturated in Photshop in order to facilitate observation of MO and membrane GFP co-expression (arrowhead). f: An image of eye-targeted co-electroporation illustrating the extent of co-electroporation and the sizes of MO and DNA electroporated regions. g: Frontal section of a MO/GFP co-electroporated embryo showing that GFP can be used to trace the axons of electroporated cells (arrowheads indicate axons at different points in their pathway). h and i: Examples of embryos electroporated with pCS2GFP in the presence (i) or absence (h) of anti-GFP MO. Morphology of the eye appeared normal in both conditions (left panel). The GFP signal was sharply reduced in the anti-GFP MO condition when analyzed 12 h after electroporation (central panels). A decrease in electroporation efficiency was not a confounding factor in this experiment as the Special Delivery lissamine-tagged MO control is efficiently loaded in both conditions (far right panel). j: Quantification of results presented in h and i (n indicates the number of embryos analyzed). Anti-GFP MO only affects expression of pCS2GFP but not of pEGFP (Clontech). k: Anti-GFP MO was co-electroporated with GFP and GAP-RFP. 48 h after electroporation, GFP and RFP fluorescence was quantified on sections and the ratio between the two calculated. (n refers to the numbers of sections quantified [3 embryos were analyzed for control and 6 for MO]). Statistical analysis: Mann-Whitney test; probabilities are indicated together with the S.E.M. l-m: Sections through an eye lipofected with GFP (green, l and m) and subsequently loaded with lissamine-tagged MOs (red) using electroporation (merge, m). n-q: Electroporated embryos can be a source of modified cells for in vitro studies. Explants and cells cultured from MO (n and o) or DNA (GFP) (p and q) electroporated embryos. Scale bars: 400 μm in h; 100 μm in a; 50 μm in d, f, and g; in 25 μm e and m; 20 μm in n; 10 μm in o.